【作者】 时亮；

【导师】 于成国；

【作者基本信息】 中国医科大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 前言 端粒酶（Telomerase）是由端粒酶RNA和蛋白质组成的核糖核蛋白质，能够催化端粒DNA部分富含串联排列的TTAGGG重复片段的合成，为染色体末端提供模板从而延伸端粒寡核苷酸片段，维持端粒长度。正常人体细胞（非生殖细胞）中端粒酶活性的表达很低，或用目前的方法几乎检测不到，而在90％以上的恶性肿瘤中可检测到端粒酶活性，相应的良性肿瘤则为阴性或仅有少数阳性。在恶性肿瘤中端粒酶的激活是细胞增殖失控的关键，从而维持端粒长度阻止细胞死亡。端粒酶与细胞生长、繁殖、衰老及肿瘤的发生密切相关，其活性有助于肿瘤的临床诊断、疗效考核和预后判断。近年研究表明，对端粒酶活性调控存在着多种不同的途径。卵巢主要合成和分泌两种女性激素，即雌激素和孕激素，其生理状态在激素的影响下呈周期性改变，激素与卵巢癌的发生、发展有密切的关系。为了研究是否存在控制端粒酶活性的激素依赖机制，本研究采用发病率很高的妇科恶性肿瘤—卵巢癌CAOV3细胞系作为靶细胞，采用TRAP-银染法检测雌二醇（Estrogen，E₂）和孕酮（Progesterone，P）对卵巢癌细胞端粒酶活性的影响，更进一步的分析激素控制端粒酶在细胞衰老中作用的分子机制和激素依赖肿瘤的癌变过程，为探索以端粒酶为靶点治疗恶性肿瘤方法提供一些参考。 实验材料与方法 1．实验材料 1．1 细胞和组织：卵巢癌CAOV3细胞系由中国医科大学细胞生物教研室提供，常规培养；卵巢癌组织由沈阳陆军总院外科手术摘除并经病理学诊断证实，置于一70℃冻存。 1．2 主要试剂：17p一雌二醇、6＊＊RP S与＊＊＊3F(美国Sma公司人孕激素（仙居制药厂厂PCR试剂盒（日本Takara公司X其它常规试剂均为国产分析纯。 2．实验方法 2．1 细胞增殖的测定仲Th法入将CAOV3细胞株单细胞悬液接种于 24孔板中，将含有不同浓度的门 p一雌二醇及孕激素的培养液加人各孔细胞中，继续培养48小时后，用酶标光度计在波长为580urn时测定每孔的吸光度K）值，确定不同浓度药物对细胞的影响趋势。 2．2 细胞端粒酶制备：将培养的细胞实验组加人含不同浓度的17p一雌二醇和孕激素的培养液，对照组加同实验组等量的乙醇门 人分别培养12h上4h／8h。收集细胞、裂解、离心、移取上清部分样品用于蛋白定量，其余迅速冷冻，－70℃保存。 2．3 组织端粒酶的制备：将肿瘤组织用高压灭菌的眼科剪刀剪碎、制勾浆、裂解，16，000转离心30分钟，移取部分上清用于蛋白定量，其余迅速冷冻，－70℃保存。用不同浓度的雌激素分别处理卵巢癌组织的端粒酶提取液 Zh／h在h，加乙醇提纯，去乙醇，放置一20℃冰箱待用。 2．4 TRAP反应；建立TRAP PCR体系50pl，扩增30个循环；最后72℃10分钟终止。 2．5 PCR产物纯化：用等体积酚和氯仿：异戊醇口4（）提纯细胞和组织提取物。 2．6 电泳与染色：在12％非变性聚丙烯酸胺凝胶上电泳，电泳缓冲液为 0．5 M X TBE。再固定，染色，还原，直到条带显色满意时用 5％乙酸终止反应，拍照或制成薄膜保存． 2．7 采用SPSS软件做t检验检测各不同浓度实验组与对照组间的差异，所有参数均用X。S表示。 ·2· 结 果 1．MTh比色法检测E。与P对CAOV3细胞增殖的影响。当E。浓度为lxlo、M时，对细胞生长有相对明显的激活效应，与对照组比较，差异显著河＜0刀5人随着浓度的增加入值呈现明显下降趋势，显示剂量一效应相关关系。P对CAOV3细胞的生长有一定的抑制作用，呈现剂量依赖效应，P浓度为 lx 10hM时作用48 h后，只有 60－89％的卵巢癌细胞存活。 2．CAOV3细胞经雌激素作用后，端粒酶活性有不同程度的上升。以薄层扫描仪分析各条带峰值总面积代表端粒酶活性，用InM、lx 10‘nM、IX 10’nM、IX 10’nM 17p一雌二醇处理 12 ／J’时＼24小时／8小时后CAOV3细胞端粒酶活性分别上升至对照组的48％（InM，12h），62叽（lx10’nM，12h）、56％ （In，24h），76．8％门 x 10’nM，24h）、53％（InM，48h）和的呢（lx 10‘nM，48h）。 3．孕激素对CAOV3细胞端粒酶活性的作用与作用时间相关。用h ＊ 10‘nM＊ 10、M孕激素处理 12 ／J’时在4 ／j＼时和48 ／J’时，可见端粒酶活性 12 ／J’时上升至对照组的 12．5％（InM，）、43．2畅（IX 10‘nM）、25％（IX 10znM）；24 ／J’时处理上升至对照组 51．1％（InM）、63．6阮（lx 10‘nM）、43．2锡(lx10、M厂48／J’时后端粒酶活性下降至对照组 10％(In入30．7％（lxlo‘nM）、38．6阮（lxlo‘nM）。 4．CAOV3细胞经同浓度的雌、孕激素混合作用后，端粒酶活性有了更显著的改变。处理组相对于标准DNASI hP－587hP之间清晰可见端粒酶条带(间距＞6hP人用 InM上 10bM的雌、孕激素混合液分别处理12小时、24小时／8小时J72小时，可见端粒酶活性在12小时在4小时非常显著的被激活，48小时后明显减弱。更多还原

【Abstract】 Telomerase is a celler reverse transcriptase which catalyzes the synthesis and extension of telomeric DNA. Telomerase activation is not observed in normal tissues of somatic origin but in almost 90% of human cancer, with the result the telomere shorten progressively with cell division and thus is a critical step for multistep carcinogenesis. The factors and mechanism involved in telomerase regulation are not well understood.Ovary is the main organ to synthesize and excrete estrogen and progesterone, which can affect its physiological change periodically. Ovary tumor is one of the tumors of the women to see frenquently. Sex steroid hormones involve in the tight regulation of telomerase expression and activity in ovary malignaint tumor. In order to find the evident that sex steroid hormones may be good candidates as physiological regulator of telomerase activity, we detected the telomerase activity of treated cells and issues by TRAP - Sliver Stain method to investigate the relationship between 17β-Estrogen, progesterone and the expression levels of telomerase activity in ovarian cancer cells and tissue and observed the morphological and functional changes of the cells. In thisstudy, we provide evidence for a role of estrogens and progesterone in the activation of the telomerase. Those results may help elucidate the mechanisms of hormonal control of telomerase activity and aid understanding of the roles of sex steroids in cellular senescence and aging as well as estrogen - induced carcinogenesis.Material and methodMaterial:Cell lines and Tissue; CAOV3 cells were purchased from Cell Biology department in China Medical University and were grown in RPMI -1640 with 10% FCS in the presence of 5% CO2 at 37℃; Ovary tumor samples were obtained from surgery of the Main Army Hospital in Shen Yang with histological confirmed Ovary Tumor. Chemical: 17β - Estrogen, Charps and Aebsf was purchased from Sigma company in USA. Progesterone was purchased from Xianju Pharmaceutical Factory in China. PCR kit, from Takara company in Japan. All other chemicals were of reagent grade.Method:1. MTT Assay: For determining the influence of the medium containing 17β -Estrogen or progesterone on cells growth, CAOV3 were treated with them for 48h and then determined in MTT Assay.2. Preparation of crude cell extracts; The cultured cells were treated with different medium containing 17β - E2 and/or progesterone of different concentration. After 12h,24h,48h,72h, the treated cells were collected and the extracts from CAOV3 was prepared by the Chaps - lysis method. Protein concentration were determined by folin method and the rest extracts were conserved in -70℃ quickly.3. Preparation of crude tiss ue extracts: The tumor samples were cut, whetted, and prepared by the Chaps -lysis method. Protein concentration were determined by folin method. The rest extracts were conserved in -70℃ quickly. The extracts were treated with 17β -Estrogen in different concentration, then purified by ethanol and reserved in -20℃.4. TRAP Assay: The reaction mixture contained the total of 50 ul, run in 30 cycles and stopped at 72℃ for 10 min.5. Purification of the amplified PCR product; To purify the PCR product of cells and tissue of ovary tumor with hydroxybenzene and chloroform: isoamyl alcohol ( 24:1).6. Electrophoresis and Sliver - Staining:In order to detect the te-lomeric repeats, PCR products were electrophoresed on a 12% polyac-rylamide gel with 0. 5M x TBE eletrophoretic buffer. Fixation, staining, and deoxidization were performed respectively and the reaction was stopped by 5% acetic acid until we have got a good show. The gel was dried and taken photographs.Finally, we contrasted the experimental groups with the control by SPSS soft.Results1. CAOV3 were treated with the medium containing 17)3 - Estrogen or progesterone for 48h and then determined in MTT Assay. We found that the growth of cells bloom obviously after treated by 10nM estrogen and subsequently decrease. Progesterone更多还原